To provide a serological detection method for the early diagnosis of reovirus disease in waterfowl,this study stably expressed the σC protein of waterfowl reovirus by BL21(DE3)prokaryotic expression system.The purified recombinant σC protein was coated with ELISA plate,and the reaction conditions were further optimized.The results showed that the optimal reaction conditions for the ELISA detection method established in this assay were as follows:antigen coating content of 1 μg/well,the best dilution of serum to be tested was 1∶50,incubation time was 45 min,the best dilution of HRP-conjugated secondary antibody was 1∶500,incubation time was 30 min,the best substrate chromogenic time was 20 min.OD450 value ≥0.3 was considered positive.This method has good specificity,sensitivity and repeatability,and has certain cross-reactivity against reovirus genotype Ⅰ and genotype Ⅱ serotypes in waterfowl.The positive rate of 105 serum samples from healthy geese was 77.33％.This study provides a sensitive and efficient method for the rapid detection of neutralizing antibodies against reovirus in waterfowl,which provides a method for the large-scale detection of clinical serum in geese,and provides a basis for the development of commercial ELISA kits.
关键词
水禽呼肠孤病毒/σC蛋白/间接ELISA方法/血清学调查
Key words
Waterfowl reovirus/Sigma C protein/Indirect ELISA method/Serological investigation
维普
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